Purine inhibitors of protein kinases, G proteins and polymerases

ABSTRACT

The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

GOVERNMENT RIGHTS

[0001] This invention was made with Government support under Grant(Contract) No. DE-AC03-76SF00098 awarded by the U.S. Department ofEnergy. The Government has certain rights in this invention.

FIELD OF THE INVENTION

[0002] The present invention relates to purine analogs that inhibit,inter alia, protein kinases, G-proteins and polymerases. In addition,the present invention relates to methods of using such purine analogs toinhibit protein kinases, G-proteins, polymerases and other cellularprocesses and methods of using such purine analogs to treat, forexample, cellular proliferative diseases and neurodegenerative diseases.

BACKGROUND OF THE INVENTION

[0003] Phosphorylation of serine, threonine and tyrosine residues byprotein kinases represents one of the most common post-translationalregulatory modifications of proteins. More than 200 protein kinases havebeen described, following either purification to homogeneity ormolecular cloning (see, Hunter, T. (1991), Methods Enzymol., 200:3-37;Hanks, S. K., et al. (1991), Methods Enzymol., 200:38-81; Hanks, S. K.1991), Curr. Opin. Struct. Biol., 1:369-383; and Hubbard, M. J., et al.(1993) Trends Biochem. Sci., 18:172-177). It is thought that as much as2-3% of eukaryotic genes encode protein kinases. The importance ofprotein kinases in physiological processes has stimulated an activesearch for specific inhibitors with potential pharmacological interest(see, Hidaka, H., et al. (1992), Annu. Rev. Pharmacol. Toxicol.,32:377-397). Several classes of compounds have been identified, such asstaurosporine, naphthalene sulfonamides (W7, ML-9, SC-9), isoquinolinederivatives (H-7, H-8, KN-62), sphingosine, tyrphostins and others, butin most cases these inhibitors display broad specificity. Only somepseudosubstrate autoinhibitory peptides show a high degree ofspecificity.

[0004] Cyclin-dependent kinases (CDK), in particular, have recentlyraised considerable interest in view of their essential role in theregulation of the cell division cycle (CDC) (see, Nigg, E. A. (1993),Trends in Cell Biol., 3:296-301; and Sherr, C. S. (1993), Cell,73:1059-1065). CDKs are highly conserved among eukaryotic species.Higher eukaryotic cells contain several isoforms of CDKs that becomeactivated in specific phases of the cell cycle. CDKs consist of acatalytic subunit, the prototype of which is CDC2, and a regulatorysubunit (cyclin). Six human CDK proteins have been described so far(see, Meyerson, M., et al. (1992), EMBO J, 11:2909-2917; Meyerson, M.,et al. (1994), Mol. Cell. Biol., 14:2077-2086; and Van den Heuvel, S.,et al. (1993), Science, 262:2050-2054), namely, CDK1 (also known asCDC2) and CDK2-6. With the exception of CDK3, for which the regulatorycyclin has not yet been identified, all these CDKs proteins areregulated by the transient association with one member of the cyclinfamily, i.e., cyclin A (CDC2, CDK2), B1-B3 (CDC2), D1-D3 (CDK2, CDK4,CDK5, CDK6), E (CDK2). Each step of the cell cycle is thought to beregulated by such CDK complexes: G1/S transition (CDK2/cyclin E,CDK3/unknown cyclin, CDK4/cyclin D1-D3, CDK6/cyclin D3), S phase(CDK2/cyclin A), G2 (CDC2/cyclin A), G2/M transition (CDC2/cyclins B).

[0005] CDKs are able to phosphorylate many proteins that are involved incell cycle events, including histones, lamins and tumor suppressorproteins, such as the retinoblastoma gene product pRb (see, Norbury, C.,et al., supra, Matsushime, H., et al. (1992), Cell, 71:323-334, Nigg, E.E. (1993), Curr. Opin. Cell. Biol., 5:187-193). In accordance with theircentral role in the cell cycle, enzyme activity is tightly controlled bymultiple mechanisms. Kinase activation requires complex formation withregulatory cyclin proteins as described above, followed by an activatingphosphorylation on Thr-161 in CDC2 or the corresponding Thr in the otherCDKs (see, e.g., Gould, K. L., et al. (1991), EMBO J., 10:3297-3309;Desai, D., et al. (1992), Mol. Biol. Cell, 3:571-582; Solomon, M. J., etal. (1992), Mol. Biol. Cell, 3:13-27). In addition, enzyme activity isnegatively regulated by phosphorylations at Tyr-15 and/or Thr-14 (see,e.g., Solomon, M. J., et al, supra; Gu, Y., et al. (1992), EMBO J,11:3995-4005; Krek, W., et al. (1991), EMBO J, 10:3331-3341; Norbury,C., et al. (1991), EMBO J, 10:3321-3329; Parker, L. L., et al. (1992),Proc. Natl. Acad. Sci. U.S.A., 89:2917-2921; McGowan, C. H., et al.(1993), EMBO J, 12:75-85), or by complex formation with inhibitorproteins like p16 (see, Serrano, M., et al. (1993), Nature (London),366:704-707; Kamb, A., et al. (1994), Nature (London), 264:436-440;Nobori, T., et al. (1994), Nature (London), 368:753-756), p27 (see,Polyak, K., et al. (1994), Cell, 78:59-66; Toyoshima, H., et al. (1994),Cell, 78:67-74), p28 (see, Hengst, L., et al. (1994), Proc. Natl. Acad.Sci. U.S.A., 91:5291-5295) and p21 (see, Gu, Y., et al. (1993), Nature(London), 366:707-710; Xiiong, Y., et al. (1993), Nature (London),366:701-704; Harper, J. W., et al. (1993), Cell, 75:805-816; Dulic, V.,et al. (1994), Cell, 76:1013-1023), the latter being inducible by p53.Especially noteworthy is the fact that deletions of the p16 gene werefound in over 50% of all human malignant cell lines tested (see, Kamb,A., supra, Nobori, T., et al., supra), although much less in primarytumor cells (see, Spruck III, C. H., et al. (1994), Nature (London),370:183-184), implicating p16 functions as tumor suppressor protein.Thus, both the cell growth signals transmitted through many oncogeneproducts and the growth inhibitory signals from several tumor suppressorproteins modulate the activity of CDKs. Although mutations in CDKsthemselves have not been associated with cancer, cyclin overexpressionhas been linked to tumorigenesis (see, Hunter, T., et al. (1991), Cell,66:1071-1074; Keyomarsi, K., et al. (1993), Proc. Natl. Acad. Sci.U.S.A., 90:1112-1116; Wang, T. C., et al. (1994), Nature (London),369:669-671.) Hence, CDKs are a promising target for developinginhibitors with antineoplastic effects and for the treatment ofcell-proliferative diseases.

[0006] The purine ring system is a key structural element of thesubstrates and ligands of many biosynthetic, regulatory and signaltransduction proteins including cellular kinases, G proteins andpolymerases. As such, the purine ring system has been a good startingpoint in the search for inhibitors of many biomedically significantprocesses. In fact, while purine analogs were being screened forinhibition of various protein kinases, a relatively selective inhibitor,olomoucine (FIG. 1), was identified that competitively inhibitsCDK2/cyclin A with an IC₅₀ of 7 iM (see, Vesely, J., et al., (1994) Eur.J. Biochem., 224:771-786). Although olomoucine exhibits moderateinhibitory activity and good selectivity for the CDK/cyclin proteinkinases, it would be advantageous to identify other purine analogs thathave increased affinity and specificity for protein kinases as well as Gproteins and polymerases. Quite surprisingly, the present inventionprovides such analogs.

SUMMARY OF THE INVENTION

[0007] The present invention provides (i) purine analogs that, interalia, inhibit protein kinases, G proteins and polymerases; (ii) methodsof using such purine analogs to inhibit protein kinases, G proteins,polymerases and other cellular processes; and (iii) pharmaceuticalcompositions comprising such purine analogs.

[0008] In one embodiment, the present invention provides purine analogshaving the generally formula:

[0009] or a pharmaceutically acceptable salt thereof.

[0010] In Formula I, R¹, R², R⁴ and R⁵ are independently selected andare functional groups including, but not limited to, H, C₁-C₈straight-chain, branched-chain, saturated and unsaturated alkyl, C₁-C₈straight-chain, branched-chain, saturated and unsaturated substitutedalkyl, aryl and substituted aryl.

[0011] In another embodiment, the present invention providespharmaceutical compositions comprising the purine analog compounds ofthe invention and a pharmaceutically acceptable carrier.

[0012] In another embodiment, the present invention provides a method ofinhibiting a protein selected from the group consisting of proteinkinases, G proteins and polymerases, the method comprising contactingthe protein with a purine analog of the present invention. In apreferred embodiment, the protein is a protein kinase. In an even morepreferred embodiment, the protein kinase is a cyclin-dependent kinase.In an even more preferred embodiment, the cyclin-dependent kinase is amember selected from the group consisting of CDK1 (CDC2), CDK2, CDK3,CDK4, CDK5, CDK6, CDK7 and CDK8 and, in particular, CDK1 and CDK5.

[0013] In another embodiment, the present invention provides a method oftreating a cellular proliferative disease, the method comprisingadministering to a mammal having the disease a therapeutically effectiveamount of a purine analog of the present invention.

[0014] In yet another embodiment, the present invention provides amethod of inhibiting the growth of a tumor cell, the method comprisingcontacting the tumor cell with a purine analog of the present invention.In a preferred embodiment, the tumor cell is selected from the groupconsisting of lung, colon, breast, ovarian, prostate and hepatic cells.In a preferred embodiment, the tumor cell is in a mammalian subject. Inanother preferred embodiment, the purine analog is formulated in apharmaceutically acceptable form with an excipient or carrier andadministered orally. In another embodiment, this method furthercomprising the step of observing for a reduction in the growth of atumor cell.

[0015] In still another embodiment, the present invention provides amethod of treating a neurodegenerative disease, the method comprisingadministering to a mammal having the disease a therapeutically effectiveamount of a purine analog of the present invention.

[0016] Other features, objects and advantages of the invention and itspreferred embodiments will become apparent from the detailed descriptionwhich follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 sets forth the structure of olomoucine and the numberingscheme for the purine nucleus.

[0018]FIGS. 2 and 3 illustrate the IC₅₀ for representative compoundsfrom Table 1.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

[0019] The present invention provides (i) purine analogs that, interalia, inhibit protein kinases, G proteins and polymerases; (ii) methodsof using such purine analogs to inhibit protein kinases, G proteins,polymerases and other cellular processes; and (iii) pharmaceuticalcompositions comprising such purine analogs.

[0020] A. Definitions

[0021] The term “independently selected” is used herein to indicate thatthe R groups, e.g., R¹, R², R⁴ and R⁵, can be identical or different(e.g., R¹, R² and R³ may all be substituted alkyls or R¹ and R² may be asubstituted alkyl and R³ may be an aryl, etc.).

[0022] A named R group will generally have the structure which isrecognized in the art as corresponding to R groups having that name. Forthe purposes of illustration, representative R groups as enumeratedabove are defined herein. These definitions are intended to supplementand illustrate, not preclude, the definitions known to those of skill inthe art.

[0023] The term “alkyl” is used herein to refer to a branched orunbranched, saturated or unsaturated, monovalent hydrocarbon radicalhaving from 1-12 carbons and preferably, from 1-6 carbons. When thealkyl group has from 1-6 carbon atoms, it is referred to as a “loweralkyl.” Suitable alkyl radicals include, for example, methyl, ethyl,n-propyl, i-propyl, 2-propenyl (or allyl), n-butyl, t-butyl, i-butyl (or2-methylpropyl), etc.

[0024] “Substituted alkyl” refers to alkyl as just described includingone or more functional groups such as lower alkyl, aryl, acyl, halogen(i.e., alkylhalos, e.g., CF₃), hydroxy, amino, alkoxy, alkylamino,acylamino, acyloxy, aryloxy, aryloxyalkyl, mercapto, both saturated andunsaturated cyclic hydrocarbons, heterocycles and the like. These groupsmay be attached to any carbon of the alkyl moiety.

[0025] The term “aryl” is used herein to refer to an aromaticsubstituent which may be a single aromatic ring or multiple aromaticrings which are fused together, linked covalently, or linked to a commongroup such as a methylene or ethylene moiety. The common linking groupmay also be a carbonyl as in benzophenone. The aromatic ring(s) mayinclude phenyl, naphthyl, biphenyl, diphenylmethyl and benzophenoneamong others.

[0026] The term “arylalkyl” is used herein to refer to a subset of“aryl” in which the aryl group is attached through an alkyl group asdefined herein.

[0027] “Substituted aryl” refers to aryl as just described including oneor more functional groups such as lower alkyl, acyl, halogen, alkylhalos(e.g., CF₃), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy,mercapto and both saturated and unsaturated cyclic hydrocarbons whichare fused to the aromatic ring(s), linked covalently or linked to acommon group such as a methylene or ethylene moiety. The linking groupmay also be a carbonyl such as in cyclohexyl phenyl ketone. The term“substituted aryl” encompasses “substituted arylalkyl.”

[0028] “Substituted arylalkyl” defines a subset of “substituted aryl”wherein the substituted aryl group is attached to the nucleus by analkyl group as defined herein.

[0029] The term “halogen” is used herein to refer to fluorine, bromine,chlorine and iodine atoms.

[0030] The term “hydroxy” is used herein to refer to the group —OH.

[0031] The term “amino” is used herein to refer to the group —NRR′,where R and R′ may independently be hydrogen, lower alkyl, substitutedlower alkyl, aryl, substituted aryl or acyl.

[0032] The term “alkoxy” is used herein to refer to the —OR group, whereR is a lower alkyl, substituted lower alkyl, aryl, substituted aryl,arylalkyl or substituted arylalkyl wherein the alkyl, aryl, substitutedaryl, arylalkyl and substituted arylalkyl groups are as describedherein. Suitable alkoxy radicals include, for example, methoxy, ethoxy,phenoxy, substituted phenoxy, benzyloxy, phenethyloxy, t-butoxy, etc.

[0033] The term “alkylamino” denotes secondary and tertiary amineswherein the alkyl groups may be either the same or different and mayconsist of straight or branched, saturated or unsaturated hydrocarbons.

[0034] The term “heterocyclic” is used herein to describe a monovalentgroup having a single ring or multiple condensed rings from 1-12 carbonatoms and from 1-4 heteroatoms selected from nitrogen, sulfur or oxygenwithin the ring. Such heterocycles are, for example, tetrahydrofuran,morpholine, piperidine, pyrrolidine, thiophene, pyridine, isoxazole,phthalimide, pyrazole, indole, furan, benzo-fused analogs of theserings, etc.

[0035] The term “substituted heterocyclic” as used herein describes asubset of “heterocyclic” wherein the heterocycle nucleus is substitutedwith one or more functional groups such as lower alkyl, acyl, halogen,alkylhalos (e.g., CF₃), hydroxy, amino, alkoxy, alkylamino, acylamino,acyloxy, mercapto, etc.

[0036] The term “pharmaceutically acceptable salt” refers to those saltsof compounds which retain the biological effectiveness and properties ofthe free bases and which are obtained by reaction with inorganic acidssuch as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, methanesulfonic acid, ethanesulfonic acid,ptoluenesulfonic acid, salicylic acid and the like. Pharmaceuticallyacceptable salts include, for example, alkali metal salts, such assodium and potassium, alkaline earth salts and ammonium salts.

[0037] The term “contacting” is used herein interchangeably with thefollowing: combined with, added to, mixed with, passed over, incubatedwith, flowed over, etc. Moreover, the purine compounds of presentinvention can be “administered” by any conventional method such as, forexample, parenteral, oral, topical and inhalation routes as describedherein.

[0038] “An amount sufficient” or “an effective amount” is that amount ofa given purine analog which exhibits the binding/inhibitory activity ofinterest or, which provides either a subjective relief of a symptom(s)or an objectively identifiable improvement as noted by the clinician orother qualified observer.

[0039] B. Purine Analogs

[0040] The purine ring is a key structural element of the substrates andligands of many biosynthetic, regulatory and signal transductionproteins including cellular protein kinases, G proteins and polymerases.Quite importantly, the present invention provides purine analogs whichcan be used to inhibit such proteins and, thus, many biomedicallyimportant processes. More particularly, the present invention providespurine analogs that inhibit, inter alia, protein kinases, G proteins,polymerases and other cellular processes. As such, the purine analogs ofthe present invention can be used to block cell-cycle progression,cellular proliferation, apoptosis as well as other cellular processes.The purine analogs of the present invention are active in thesubnanomolar and submicromolar ranges.

[0041] In one embodiment, the present invention provides purine analogshaving the generally formula:

[0042] or a pharmaceutically acceptable salt thereof.

[0043] In Formula I, R¹, R², R⁴ and R⁵ are independently selected andare functional groups including, but not limited to, H, C₁-C₈straight-chain, branched-chain, saturated and unsaturated alkyl, C₁-C₈straight-chain, branched-chain, saturated and unsaturated substitutedalkyl, aryl and substituted aryl.

[0044] Within the scope of the above Formula I, certain embodiments arepreferred, namely those in which R¹ and R² are independently selectedand are functional groups including, but not limited to, H, aryl,substituted aryl, C₁-C₈ straight-chain, saturated alkyl substituted witharyl and C₁-C₈ straight-chain, saturated alkyl substituted withsubstituted aryl; R³ is a functional group including, but not limitedto, C₁-C₈ branched-chain saturated alkyl and C₁-C₈ branched-chainunsaturated alkyl; and

[0045] R⁴ and R⁵ are independently selected and are functional groupsincluding, but not limited to, H, C₁-C₈ straight-chain, branched-chain,saturated and unsaturated alkyl, C₁-C₈ straight-chain, branched-chain,saturated and unsaturated substituted alkyl, aryl and substituted aryl.

[0046] In another preferred embodiment, R¹ and R² are independentlyselected and are functional groups including, but not limited to, H,unsubstituted aryl and substituted aryl; R³ is isopropyl; and R⁴ and R⁵are independently selected and are functional groups including, but notlimited to, H, C₁-C₈ saturated and unsaturated branched-chain alkyl andC₁-C₈ saturated and unsaturated branched-chain substituted alkyl.

[0047] In another preferred embodiment, R⁴ and R⁵ are independentlyselected and are functional groups including, but not limited to, H, and

[0048] wherein X is a member selected from the group consisting of H,OH, CH₂OH, C(O)NH₂, SH, COOH or a pharmaceutically acceptable saltthereof and COOR⁷, wherein R⁷ is lower alkyl; and R⁶ is a memberselected from the group consisting of H, C₁-C₈ straight-chain alkyl,C₁-C₈ branched-chain alkyl, C₁-C₈ straight-chain substituted alkyl,C₁-C₈ branched-chain substituted alkyl.

[0049] With respect to above embodiment, X is preferably COOH; and R⁶ isindependently selected and is a functional group including, but notlimited to, H, —CH₃, —(CH₂)₃NHC(═NH)NH₂, —CH₂CONH₂, —CH₂CO₂H, —CH₂SH,—(CH₂)₂CONH₂, —(CH₂)₂CO₂H, —CH₂(4-imidazoyl), —CH(CH₃)CH₂CH₂,—CH₂CH(CH₃)₂, —(CH₂)₄NH₂, —(CH₂)₂SCH₃, —CH₂Ph, —CH₂OH, —CH(CH₃)OH,—CH₂(3-indolyl), —CH₂(4-hydroxyphenyl) and —CH(CH₃)₂.

[0050] In such embodiments, R¹ and R² are independently selected and arefunctional groups including, but not limited to, H and aryl substitutedin at least one of positions 3, 4, 5 with a member independentlyselected from the group consisting of halogen, alkoxy, trihalomethyl,amino, hydroxyl, thiol, sulfonic acid, sulfonic acid, amide, ester andcarboxylic acid.

[0051] Table 1 sets forth purine compounds in accordance with thepresent invention which are particularly preferred. The compounds inthis table and throughout this specification are referred to by codenumbers, which are used for convenience only, and are strictly arbitraryfor purposes of this invention. TABLE 1 Exemplary Purine Analogs IC₅₀cdc2/cyclinB Structure Code Name (additional kinases) Class 1

NG-30 330 nM 2000 nM(CDK2/cyclinE) >33,000 nM(GSK-3) 8,000 nM(erk1)Class 2a

NG-64 290 nM

NG-65 400 nM

NG-42 4300 nM

NG-43 4300 n5M

NG-44 500 nM

NG-45 270 nM

NG-46 9000 nM

NG-47 430 nM

NG-50 2800 nM

NG-51 420 nM

NG-52 220 nM

NG-53 10,000 nM

NG-54 2700 nM Class 2b

NG-35 150 nM 140(cdk2/cylinE) 15 nM(cdk5/p25) 4500(GSK-3) 3000(erk1)

NG-76 600 nM 400(CDK2/cyclinE)

NG-75 230 nM 150(CDK2/cyclinE)

NG-33 130 nM 80 nM(CDK2/cyclinE) 20,000 nM(GSK-3) >10,000 nM(erk1)

NG-36 100 nM 100 nM(cd2/cyclinE) 13,000 nM(GSK-3) >10,000 nM(erk1) Class2c

NG-16 240 nM 180 nM(CDK2/cyclinE) 23,000 nM(GSK-3) 50,000 nM(erk1)

NG-26 330 nM 230 nM(CDK2/cyclinE) >33,000 nM(GSK-3) 33,000 nM(erk1)

NG-40 600 nM

NG-49 2800 nM Class 3

NG-60 35 nM 30 nM (CDK2/CyclinE)

NG-56 35 nM 55 nM (CDK2/CyclinE)

NG-57 400 nM

NG-59 800 nM

NG-62 500 nM

NG-95 approx. 20 nM

NG-96 approx. 30 nM

NG-97 30 nM

NG-98 30 nM

NG-94 approx. 100 nM

NG-61 2300 nM

[0052] It will be readily appreciated by those of skill in the art thatdepending on the substituents, the purine analogs of the presentinvention can be a racemic mixture or either of a pair of diastereomersor enantiomers.

[0053] The purine analogs of the present invention can be synthesized ina variety of ways, using conventional synthetic chemistry techniques.Typically, the compounds of the present invention are prepared accordingto the reaction scheme set forth in Scheme I, wherein R¹, R², R³, R⁴,and R⁵ are as defined above. The use of appropriate organic solvents,temperature and time conditions for running the reactions are within thelevel of skill in the art. Reactions of this type are generallydescribed by Norman, et al., J. Am. Chem. Soc. 118:7430-7431 (1996); andGray, et al., Tetrahedron Letters, 38:1161-1164 (1997), the teachings ofwhich are incorporated herein by reference. Moreover, suitable synthesisreactions are illustrated herein by the representative examples.Necessary starting materials can be obtained by standard procedures oforganic chemistry.

[0054] Briefly, as illustrated in Scheme I, a purine derivative with ahalogen at the 2-position is alkylated at the 9-position with an alcoholusing the Mitsonubo alkylation. Following the alkylation, the purinederivative is aminated at the 6-position with an amine. Once prepared,the purine analogs can be purified (e.g., by TLC), characterized (e.g.,by Reverse Phase HPLC) and analyzed (e.g., by high resolutionspectroscopy using, for example, ¹H NMR or FAB-MS).

[0055] C. Uses for the Purine Analogs of the Present Invention

[0056] The purine analogs of the present invention can be used either invitro or in vivo for a variety of purposes. As mentioned, the purineanalogs of the present invention can be used to inhibit protein kinases,G proteins and polymerases. Moreover, the purine analogs of the presentinvention can be used to treat cellular-proliferative diseases. Inaddition, the compounds of the present invention can be used asmolecular tools and molecular probes.

[0057] As such, in one embodiment, the present invention provides amethod of inhibiting a protein kinase, a G protein or a polymerase, themethod comprising contacting the protein kinase, the G protein or thepolymerase with a purine analog having the general formula:

[0058] or a pharmaceutically salt thereof. The prior discussionspertaining to R¹, R², R⁴, R⁵ and R⁶, their definitions and preferredembodiments are fully applicable to the purine analogs used in thismethod and, thus, will not be repeated. The protein kinase, G protein orpolymerase is contacted with the purine analog in an amount sufficientto effect inhibition.

[0059] Protein kinases which can be inhibited using the purine analogsof the present invention include, but are not limited to,cyclin-dependent kinases (CDKs), MAP kinases (p38, ERK),(MAPK/MEK/MEKK), cAMP-dependent kinase, c-GMP-dependent kinase,Calmodulin-dependent kinase, CSK (C-src like kinase) pp 60 c-src, myosinlight chain kinase, JNK kinase, IKB kinase, Protein kinase C, etc. In apresently preferred embodiment, the protein kinase is a CDK. Such CDKsinclude CDK1 (or, interchangeably, CDC2) and CDK2-CDK8. In an even morepreferred embodiment, the CDK is CDC2, CDK2 or CDK5 as many of thepurine analogs of the present invention exhibit increased affinity andspecificity with respect to these CDKs. G proteins can be inhibitedusing the compounds of the present invention include, but are notlimited to, GTP binding proteins. Polymerases which can be inhibitedusing the purine analogs of the present invention include, but are notlimited to, DNA polymerase á, DNA polymerase ó, DNA topoisomerase I,topoisomerase II, phosphatases, telomerases, etc. Other protein kinases,G proteins and polymerases which can be inhibited using the purineanalogs of the present invention will be known to those of skill in theart.

[0060] Purine analogs of the present invention suitable for use ininhibiting protein kinases, G proteins or polymerase can readily beidentified using in vitro and in vivo screening assays. For instance,purine analogs having protein kinase inhibitory activity can be screenedfor using the CDK2/CYCLIN A microtiter-based solution-phase proteinkinase assay described in the examples and by Buxbaum, J. D., et al.Anal. Biochem. 1988, 169:209-215. Similarly, numerous assays exist whichcan be used to screen a given purine analog for G protein inhibitoryactivity or polymerase inhibitory activity. Such assays are described,for example, by Vesley, J., et al, Eur. J. Biochem., 1994, 224:771-786,the teachings of which are incorporated herein by reference. Otherassays known to and used by those of skill in the art can also be usedto screen a given purine analog for inhibitory properties againstprotein kinases, G proteins and polymerases.

[0061] In addition, the purine analogs of the present invention can beused to treat cellular-proliferative disease, the method comprisingadministering to a mammalian subject having a cellular-proliferativedisease a therapeutically effective amount of a purine analog having thegeneral formula:

[0062] or a pharmaceutically salt thereof. The prior discussionspertaining to R¹, R², R⁴, R⁵ and R⁶, their definitions and preferredembodiments are fully applicable to the purine analogs used in thismethod and, thus, will not be repeated.

[0063] Cellular proliferative diseases which can be treated using thepurine analogs of the present invention include, but are not limited to,abnormal stimulation of endothelial cells (e.g., atherosclerosis), solidtumors and tumor metastasis, benign tumors, for example, hemangiomas,acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas,vascular malfunctions, abnormal wound healing, inflammatory and immunedisorders, Bechet's disease, gout or gouty arthritis, abnormalangiogenesis accompanying, for example, rheumatoid arthritis, psoriasis,diabetic retinopathy, other ocular angiogenic diseases such asretinopathy of prematurity (retrolental fibroplastic), maculardegeneration, corneal graft rejection, neuroscular glaucoma and OsterWebber syndrome, psoriasis, restinosis, fungal, parasitic and viralinfections such cytomegaloviral infections.

[0064] In a preferred embodiment, the present invention provides amethod of inhibiting the growth of a tumor cell, the method comprisingcontacting the tumor cell with a compound having the general formula:

[0065] or a pharmaceutically acceptable salt thereof. The priordiscussions pertaining to R¹, R², R⁴, R⁵ and R⁶, their definitions andpreferred embodiments are fully applicable to the purine analogs used inthis method and, thus, will not be repeated.

[0066] In accordance with the above method, tumor cells include, but arenot limited to, lung, colon, breast, ovarian, prostate and hepatic tumorcells as well as squamous cell carcinomas. In a presently preferredembodiment, the tumor cells are present in a mammalian subject.Mammalian subjects include, but are not limited to, humans, laboratoryanimals, domestic pets and farm animals. In a further preferredembodiment, the above method further comprises the step of observing fora reduction in the growth of the tumor cells.

[0067] In another embodiment, the present invention provides a method oftreating cancer, the method comprising administering to a mammaliansubject having cancer a therapeutically effective amount of a compoundhaving the general formula:

[0068] or a pharmaceutically acceptable salt thereof. The priordiscussions pertaining to R¹, R², R⁴, R⁵ and R⁶, their definitions andpreferred embodiments are fully applicable to the purine analogs used inthis method and, thus, will not be repeated.

[0069] The compounds of the present invention are useful for treating awide variety of cancers. Such cancers include, by way of example and notlimitation, carcinomas such as pharynx, colon, rectal, pancreatic,stomach, liver, lung, breast, skin, prostate, ovary, cervical, uterineand bladder cancers; leukemias; lymphomas; gliomas; retinoblastomas; andsarcomas. Moreover, in accordance with the above method, mammaliansubjects include, but are not limited to, humans, laboratory animals,domestic pets and farm animals.

[0070] Purine analogs suitable for use in the methods of the presentinvention can readily be identified using in vitro and in vivo screeningassays. Such assays may screen for the ability of a particular compoundto inhibit malignant tumor cell growth or to abolish tumorigenicity ofmalignant cells in vitro or in vivo. For instance, tumor cell lines canbe exposed to varying concentrations of a purine analog of interest, andthe viability of the cells can be measured at set time points using thealamar Blue® assay (commercially available from BioSource, Internationalof Camarillo, Calif.). When alamar Blue dye is added to the culturemedium, the dye is reduced by cellular mitochondrial enzymes yielding asoluble product with substantially enhanced fluorescence. Thisfluorescence can be measured with a fluorimeter, whereby the signal isdirectly proportional to the cell number. Using this information, IC₅₀(concentration of compound lethal to 50% of a cell culture as comparedto a control culture) values for the compounds of interest can bereadily be calculated.

[0071] As will be appreciated by the skilled artisan, many varieties ofmalignant tumor cell cultures and cell lines can be used to screen foractivity, including but not limited to MDA MB 231 (breast), MCF-7(breast), MDA MB 468 (breast), Siha (squamous cell carcinoma), A549(non-small cell lung), HL-60 (leukemia) Ovcar-3 (ovarian), etc. Inaddition, the purine analogs of the present invention can be screened pmthe National Cancer Institute panel of 60 human tumor cell lines. Ofcourse, other in vitro and/or in vivo assays to screen for anti-tumorand/or anti-cancer activity known to and used by the skilled artisan canalso be employed to identify effective purine analogs useful in themethods of the present invention.

[0072] In another preferred embodiment, the purine analogs of thepresent invention can be used to treat a neurodegenerative disease, themethod comprising administering to a mammal having such a disease atherapeutically effective amount of a compound having the generalformula:

[0073] or a pharmaceutically acceptable salt thereof. The priordiscussions pertaining to R¹, R², R⁴, R⁵ and R⁶, their definitions andpreferred embodiments are fully applicable to the purine analogs used inthis method and, thus, will not be repeated.

[0074] Neurodegenerative diseases which can be treated using the purineanalog compounds of the present invention include, but are not limitedto, neurodegenerative pathologies involving multiple neuronal systemsand/or brainstem including Alzheimer's disease, AIDS-related dementia,Leigh's disease, diffuse Lewy body disease, epilepsy, Multiple systematrophy, Guillain-Barre syndrome, lysosomal storage disorders such aslipofuscinosis, late-degenerative stages of Down's syndrome, Alper'sdisease, vertigo as result of CNS degeneration, etc. Otherneurodegenerative diseases which can be treated using the purine analogsof the present invention will be readily apparent to those of skill inthe art.

[0075] In addition, in view of their cell-cycle arresting activities,the purine analogs of the present invention can be used to inhibitundesirable proliferation, including, as described above, cancer,psoriasis, growth of fungi, parasites, viruses, plants, etc. Moreover,the purine analogs of the present invention have apoptosis-inducingeffects in actively dividing cells and, thus, this property can beadvantageously used to treat various disease states associated withundesirable proliferation. Such uses are described, for example, inMeijer, L., Trends in Cell Biology (1986), 6:393-397, the teachings ofwhich are incorporated herein by reference for all purposes.

[0076] In addition to the foregoing, the purine analogs of the presentinvention can be used in vitro as molecular tools and probes. Forinstance, since CDK inhibitors arrest cells both in G1 and late G2/earlyprophase, they can be used to synchronize cells when used preferably incombination with another synchronizing agent/method (e.g., when used incombination with aphidicolin). Moreover, the purine analogs of Inaddition, immobilized CDK inhibitors can be used for affinitypurification/depletion of CDKs from cellular extracts. Such purineanalogs will be particularly useful for massive purification ofexpressed CDKs (for crystallography or screening purposes). In addition,such purine analogs are useful for comparative analysis of CDKsextracted from cells at difference developmental or cell-cycle stages(variation of concentration, kinase activity, post-translationalmodifications, etc.).

[0077] D. Pharmaceutical Formulations/Routes of Administration

[0078] The compounds, i.e., purine analogs, of the present invention canbe administered to a mammal, e.g., a human patient, alone, in the formof a pharmaceutically acceptable salt, or in the form of apharmaceutical composition where the compound is mixed with suitablecarriers or excipient(s) in a therapeutically effective amount, e.g., atdoses effective to inhibit a protein kinase or a cellular process orachieve amelioration of symptoms of a disease associated with a proteinkinase.

[0079] The compounds of this invention can be incorporated into avariety of formulations for therapeutic administration. Moreparticularly, the compounds of the present invention can be formulatedinto pharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, pills, powders, granules, dragees, gels, slurries,ointments, solutions, suppositories, injections, inhalants and aerosols.As such, administration of the compounds can be achieved in variousways, including oral, buccal, rectal, parenteral, intraperitoneal,intradermal, transdermal, intracheal, etc., administration. Moreover,the compound can be administered in a local rather than systemic manner,for example via injection of the compound directly into a solid tumor,often in a depot or sustained release formulation. In addition, thecompounds can be administered in a targeted drug delivery system, forexample, in a liposome coated with tumor-specific antibody. Suchliposomes will be targeted to and taken up selectively by the tumor.

[0080] The purine analogs of the present invention can be administeredalone, in combination with each other, or they can be used incombination with other known compounds (e.g., other drugs, such asanti-cancer drugs (e.g., AZT), anti-mitotics, anti-inflammatories,antibiotics, corticosteroids, vitamins, etc.). More particularly, thecompound of the present invention can be used in conjunctive therapywith other known chemotherapeutic or antineoplastic agents (e.g., vincaalkaloids, antibiotics, antimetabolites, platinum coordinationcomplexes, etc.). For instance, the compounds of the present inventioncan be used in conjunctive therapy with a vinca alkaloid compound, suchas vinblastine, vincristine, taxol, etc.; an antibiotic, such asadriamycin (doxorubicin), dactinomycin (actinomycin D), daunorubicin(daunomycin, rubidomycin), bleomycin, plicamycin (mithramycin) andmitomycin (mitomycin C), etc.; an antimetabolite, such as methotrexate,cytarabine (AraC), azauridine, azaribine, fluorodeoxyuridine,deoxycoformycin, mercaptopurine, etc.; or a platinum coordinationcomplex, such as cisplatin (cis-DDP), carboplatin, etc. In addition,those of skill in the art will appreciate that the compounds of thepresent invention can be used in conjunctive therapy with other knownchemotherapeutic or antineoplastic compounds. In pharmaceutical dosageforms, the compounds may be administered in the form of theirpharmaceutically acceptable salts, or they may also be used alone or inappropriate association, as well as in combination with otherpharmaceutically active compounds.

[0081] Suitable formulations for use in the present invention are foundin Remington's Pharmaceutical Sciences (Mack Publishing Company,Philadelphia, Pa., 17th ed. (1985)), which is incorporated herein byreference. Moreover, for a brief review of methods for drug delivery,see, Langer, Science 249:1527-1533 (1990), which is incorporated hereinby reference. The pharmaceutical compositions described herein can bemanufactured in a manner that is known to those of skill in the art,i.e., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes. The following methods and excipients are merelyexemplary and are in no way limiting.

[0082] For injection, the compounds can be formulated into preparationsby dissolving, suspending or emulsifying them in an aqueous ornonaqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives. Preferably, the compounds of theinvention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiological saline buffer. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart.

[0083] For oral administration, the compounds can be formulated readilyby combining with pharmaceutically acceptable carriers that are wellknown in the art. Such carriers enable the compounds to be formulated astablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilicsuspensions, liquids, gels, syrups, slurries, suspensions and the like,for oral ingestion by a patient to be treated. Pharmaceuticalpreparations for oral use can be obtained by mixing the compounds with asolid excipient, optionally grinding a resulting mixture, and processingthe mixture of granules, after adding suitable auxiliaries, if desired,to obtain tablets or dragee cores. Suitable excipients are, inparticular, fillers such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

[0084] Dragee cores are provided with suitable coatings. For thispurpose, concentrated sugar solutions may be used, which may optionallycontain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,polyethylene glycol, and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. Dyestuffs or pigments maybe added to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

[0085] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

[0086] For buccal administration, the compositions may take the form oftablets or lozenges formulated in conventional manner.

[0087] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas, or from propellant-free, dry-powder inhalers. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

[0088] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

[0089] Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

[0090] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter, carbowaxes, polyethylene glycolsor other glycerides, all of which melt at body temperature, yet aresolidified at room temperature.

[0091] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

[0092] Alternatively, other delivery systems for hydrophobicpharmaceutical compounds may be employed. Liposomes and emulsions arewell known examples of delivery vehicles or carriers for hydrophobicdrugs. Certain organic solvents such as dimethylsulfoxide also may beemployed, although usually at the cost of greater toxicity.Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various types of sustained-releasematerials have been established and are well known by those skilled inthe art. Sustained-release capsules may, depending on their chemicalnature, release the compounds for a few weeks up to over 100 days.

[0093] The pharmaceutical compositions also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include but are not limited to calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols.

[0094] Pharmaceutical compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in a therapeutically effective amount. The amount ofcomposition administered will, of course, be dependent on the subjectbeing treated, on the subject's weight, the severity of the affliction,the manner of administration and the judgment of the prescribingphysician. Determination of an effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

[0095] For any compound used in the method of the invention, atherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes the IC₅₀ asdetermined in cell culture (i.e., the concentration of test compoundthat is lethal to 50% of a cell culture), or the IC₁₀₀ as determined incell culture (i.e., the concentration of compound that is lethal to 100%of a cell culture). Such information can be used to more accuratelydetermine useful doses in humans. Initial dosages can also be estimatedfrom in vitro or in vivo data.

[0096] Initial dosages can also be formulated by comparing theeffectiveness of the compounds described herein in cell culture assayswith the effectiveness of known drugs. For instance, when use asanticancer agents, initial dosages can be formulated by comparing theeffectiveness of the compounds described herein in cell culture assayswith the effectiveness of known anticancer drugs such as vincristine. Inthis method, an initial dosage can be obtained by multiplying the ratioof effective concentrations obtained in cell culture assay for the acompound of the present invention and a known anti-cancer drug by theeffective dosage of the known anti-cancer drug. For example, if acompound of the present invention is twice as effective in cell cultureassay than vincristine (i.e., the IC₅₀ of that compound is equal toone-half the IC₅₀ of vincristine in the same assay), an initialeffective dosage of the compound of the present invention would beone-half the known dosage for vincristine. Using these initialguidelines one having ordinary skill in the art could determine aneffective dosage in humans.

[0097] Moreover, toxicity and therapeutic efficacy of the compoundsdescribed herein can be determined by standard pharmaceutical proceduresin cell cultures or experimental animals, e.g., by determining the LD₅₀,(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index and can beexpressed as the ratio between LD₅₀ and ED₅₀. Compounds which exhibithigh therapeutic indices are preferred. The data obtained from thesecell culture assays and animal studies can be used in formulating adosage range that is not toxic for use in human. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See, e.g., Fingl et al., 1975, In: ThePharmacological Basis of Therapeutics, Ch. 1, p. 1).

[0098] Dosage amount and interval may be adjusted individually toprovide plasma levels of the active compound which are sufficient tomaintain therapeutic effect. Usual patient dosages for oraladministration range from about 50-2000 mg/kg/day, commonly from about100-1000 mg/kg/day, preferably from about 150-700 mg/kg/day and mostpreferably from about 250-500 mg/kg/day. Preferably, therapeuticallyeffective serum levels will be achieved by administering multiple doseseach day. In cases of local administration or selective uptake, theeffective local concentration of the drug may not be related to plasmaconcentration. One having skill in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

[0099] The invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes, and are not intended to limit the invention in any manner.Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

EXAMPLES

[0100] Example 1 illustrates a general synthetic scheme for producingthe purine derivatives of the invention on a solid support. Thesolid-phase synthesis strategy exemplified by Scheme 2 involvesattaching the growing compound to the solid-support via the side-chainat position 2 of the purine ring structure.

Example 2

[0101] Example 2 illustrates a generalized synthetic route to purinederivatives on a solid support. The solid-phase synthesis strategyexemplified by Scheme 2 involves attaching the growing compound to thesolid-support via the side-chain at position 9 of the purine ringstructure.

Example 3

[0102] Example 3 illustrates a general route to purine derivativessynthesized on a solid support. The route exemplified by Scheme 3involves attaching the growing compound to the solid-support via thesubstituent at the 6-position of the purine ring.

Example 4

[0103] Example 4 details the alkylation of position 9 of a purinenucleus. The synthetic route is summarized in Scheme 4.

[0104] 2-Fluoro-6-chloropurine (900 mg 5.20 mmol) and PPh3 (3.0 g, 10.4mmol) were combined in a flame-dried flask under N₂. Freshly distilledTHF (60 mL) was added followed by 2-propanol (800 iL, 10.4 mmol). Themixture was cooled to −10° C. in an ethylene glycol/dry ice bath. DEAD(850 iL, 10.4 mmol) was added over 10 min. The mixture was stirred at−10° C. and gradually returned to room temperature over 3 hours.

[0105] The reaction was quenched by adding water (500 iL) to thereaction mixture. The solvent was removed in vacuo to viscous yellowoil. The oil was azeotroped with CH₂Cl₂ (2×10 mL) to remove trace THF.Purification was effected by column chromatography on silica gel elutedwith CH₂Cl₂. The CH₂Cl₂ was removed from the desired fraction. Thedesired product was isolated in 57% yield as a white powder.

Example 5

[0106] Example 5 illustrates the synthetic route to animation of the

[0107] The compound from Example 4 (3.75 g, 17.47 mmol) was combinedwith 3-chloroaniline (1.85 mL, 17.47 mmol) and diisopropylethylamine(3.05 mL, 17.47 mmol) in n-BuOH. The reaction mixture was heated to 70°C.-80° C. for 11 hours. The n-BuOH was removed under vacuum and theresulting residue was suspended in H₂O to produce a slurry. The productwas isolated by filtration, washed with small portions of CH₂Cl₂ andEt₂O. The product was dried first under a stream of air and then undervacuum. The desired product was isolated in 58% yield.

Example 6

[0108] Example 6 details the amination of the 2-position of the purinering system. The synthetic route is illustrated in Scheme 6.

[0109] The compound from Example 5 (1.55 g, 5.10 mmol),2-amino-3-methyl-1-butanol (559 iL, 5.10 mmol) and diisopropylethylamine(892 iL, 5.10 mmol) were combined in n-BuOH. The mixture was heated toapproximately 100° C. The solvent was removed under reduced pressure andthe residue purified by silica gel chromatography using 99:1CH₂Cl₂:MeOH. The desired product was isolated in 71% yield.

[0110] The above examples illustrate both general and specific methodsfor synthesizing a wide array of the purine derivatives of the presentinvention.

Example 7

[0111] This example illustrates a CDK2/CYCLIN A Microtiter ProteinKinase Assay which can be used to screen the purine analogs of thepresent invention for inhibitory activity.

[0112] 1. Required buffers and solutions

[0113] a. Buffer A: 80 mM Tris (pH=7.2) mM MgCl₂

[0114] Recipe: 4.84 g Tris (F.W.=121.1 g/mol) 4.07 g MgCl₂ (F.W.=203.31g/mol) dissolved in 500 mL of ddH₂O. pH adjusted to 7.2 with HCl.

[0115] b. Histone Hl solution: 0.45 mg/ml Histone Hl in 20 mM HEPESpH=72.

[0116] Recipe: 5 mg of Histone Hl in 11.111 mL of 20 mM HEPES pH=7.2.477 mg of HEPES PROVIDED IN 1 mL ALIQUOTS. Store at −80° C.

[0117] C. ATP solution: 90 iM ATP, 300 ig/mL BSA, 3 mM DTT.

[0118] Recipe: 9.25 mg DTT, 1.01 mg ATP (F.W.=560 g/mol), 6 mg BSAdissolved in 20 mL ddH₂O. PROVIDED IN 1 mL ALIQUOTS. Store at −80° C.

[0119] d. CDK2 solution: 10 mM HEPES pH=7.2, 25 mM NaCl, 0.5 mM DTT, 10%glycerol. PROVIDED IN 192 iL ALIQUOTS. Store at −80° C.

[0120] 2. Stepwise Description of Assay.

[0121] a. Prepare solutions of inhibitors at three times the desiredfinal assay concentration in ddH₂O 15% DMSO by volume.

[0122] b. Dispense 20 iL of inhibitors to the well of amicrotiter-formatted assay tray.

[0123] C. Thaw Histone Hl solution (1 mL aliquot), ATP solution (1 mLaliquot) and CDK2 solution (192 iL aliquot).

[0124] d. Dilute 192 iL of CDK2 solution into 2.1 mL of buffer A. Swirlto mix. Dispense 20 iL of this solution to each well using amultichannel pipetman. (Note it is important to have a fairly pointedtrough for loading the multichannel to avoid running out of solution.)

[0125] e. Mix 1 mL of Histone Hl solution with 1 mL of the ATP solutionin a 10 mL screw cap tube. Swirl to mix. Add 2-3 iL (Depending on howfresh you hot ATP is) of ã-³²P-ATP (10 iCi/mL). Mix thoroughly to geteven distribution of hot ATP but watch out for BSA frothing! Dispense towells with multichannel pipetman; mix the solution in the wells half adozen times with the multichannel pipetman.

[0126] f. Let reactions proceed for 30 minutes. While reactions arerunning you can

[0127] i) Presoak a 9×12 cm piece of nitrocellulose (0.22 Micron) inwater for 10 minutes.

[0128] ii) Load the nitrocellulose paper onto the dot blot. Screw thefour clamps down (do this in a diagonal fashion) till they are fingertight. Then apply a vacuum and continue to tighten the screws until theyare tight. Turn off the vacuum and load 100 iL of water into each wellof the dot blot to rehydrate the membrane. Apply a weak vacuum to removethe excess water, but be careful not to dry out the membrane.

[0129] iii) Just before the 30 minutes is up add 35 iL of 10% TCA toeach well of the dot blot.

[0130] g. Using the multichannel pipetman transfer 35 iL of the reactionmixtures to each well of the dot blot in the same fashion as the ATP wasdispensed (to insure equal reaction times).

[0131] h. Add an additional 35 iL of 10% TCA and apply a weak vacuumuntil the wells are free of liquid. Repeat the process of adding 35 iLof 10% TCA and draining with the vacuum two more times. Remember to turnoff vacuum when the wells are free of liquid or when you are refilling.

[0132] i. Add 35 iL of water to each well of the dot blot and apply aweak vacuum until the wells are free of liquid. Carry out this process atotal of four times.

[0133] j. Transfer the nitrocellulose membrane from the dot blotapparatus into a small tray containing enough water to cover themembrane. Let the membrane sit in the water for ten minutes then decant.Wash the membrane in this fashion with three batches of water.

[0134] k. Let the membrane dry completely before analysis with thephosphoimager.

[0135] It is to be understood that the above description is intended tobe illustrative and not restrictive. Many embodiments will be apparentto those of skill in the art upon reading the above description. Thescope of the invention should, therefore, be determined not withreference to the above description, but should instead be determinedwith reference to the appended claims, along with the full scope ofequivalents to which such claims are entitled. The disclosures of allarticles and references, including patent applications and publications,are incorporated herein by reference for all purpose.

What is claimed is:
 1. A compound having the formula.

wherein, R¹, R², R, R⁴ and R⁵ are independently members selected fromthe group consisting of H, C₁-C₈ straight-chain, branched-chain,saturated and unsaturated alkyl, C₁-C₈ straight-chain, branched-chain,saturated and unsaturated substituted alkyl, aryl and substituted aryl.2. A compound according to claim 1, wherein R¹ and R² are independentlymembers selected from the group consisting of H, aryl, substituted aryl,C₁-C₈ straight-chain, saturated alkyl substituted with aryl and C₁-C₈straight-chain, saturated alkyl substituted with substituted aryl; R³ isa member selected from the group consisting of C₁-C₈ branched-chainsaturated alkyl and C₁-C₈ branched-chain unsaturated alkyl; and R⁴ andR⁵ are independently members selected from the group consisting of H,C₁-C₈ straight-chain, branched-chain, saturated and unsaturated alkyl,C₁-C₈ straight-chain, branched-chain, saturated and unsaturatedsubstituted alkyl, aryl and substituted aryl.
 3. A compound according toclaim 2, wherein R¹and R² are independently members selected from H,unsubstituted aryl and substituted aryl; R³ is isopropyl; and R⁴ and R⁵are independently members selected from the group consisting of H, C₁-C₈saturated and unsaturated branched-chain alkyl and C₁-C₈ saturated andunsaturated branched-chain substituted alkyl.
 4. A compound according toclaim 3, wherein R⁴ and R⁵ are independently members selected from thegroup consisting of H, and

 wherein X is a member selected from the group consisting of H, OH,CH₂OH, C(O)NH₂, SH, COOH or a pharmaceutically acceptable salt thereofand COOR⁷, wherein R⁷ is lower alkyl; and R⁶ is a member selected fromthe group consisting of H, C₁-C₈ straight-chain alkyl, C₁-C₈branched-chain alkyl, C₁-C₈ straight-chain substituted alkyl, C₁-C₈branched-chain substituted alkyl.
 5. A compound according to claim 4,wherein X is COOH; and R⁶ is a member independently selected from thegroup consisting of H, —CH₃, —(CH₂)₃NHC(═NH)NH₂, —CH₂CONH₂, —CH₂CO₂H,—CH₂SH, —(CH₂)₂CONH₂, —(CH₂)₂CO₂H, —CH₂(4-imidazoyl), —CH(CH₃)CH₂CH₂,—CH₂CH(CH₃)₂, —(CH₂)₄NH₂, —(CH₂)₂SCH₃, —CH₂Ph, —CH₂OH, —CH(CH₃)OH,—CH₂(3-indolyl), —CH₂(4-hydroxyphenyl) and —CH(CH₃)₂.
 6. A compoundaccording to claim 4, wherein R¹and R² are independently membersselected from the group consisting of H and aryl substituted in at leastone of positions 3, 4, 5 with a member independently selected from thegroup consisting of halogen, alkoxy, trihalomethyl, amino, hydroxyl,thiol, sulfonic acid, sulfonic acid, amide, ester and carboxylic acid.7. A compound according to claim 6, having the structure

wherein R⁸ is a member selected from the group consisting of H,substituted benzyl groups, substituted C₁-C₃ straight chain alkyl group,heterocycles and substituted heterocycles.
 8. A compound according toclaim 6, wherein said aryl group is a member selected from the groupconsisting of polycyclic aromatic hydrocarbons and substitutedpolycyclic aromatic hydrocarbons.
 9. A compound according to claim 1having a structure selected from the group consisting of


10. A pharmaceutical composition comprising a compound in accordancewith claim 1 and a pharmaceutically acceptable carrier.
 11. Apharmaceutical composition comprising a compound in accordance withclaim 9 and a pharmaceutically acceptable carrier.
 12. A method ofinhibiting a protein selected from the group consisting of proteinkinases, G proteins and polymerases, said method comprising contactingsaid protein with a compound of claim
 1. 13. A method in accordance withclaim 12, wherein said protein is a protein kinase.
 14. A method inaccordance with claim 13, wherein said protein kinase is acyclin-dependent kinase.
 15. A method in accordance with claim 14,wherein said cyclin-dependent kinase is a member selected from the groupconsisting of CDK1 (CDC2), CDK2, CDK3, CDK4, CDK5, CDK6, CDK7 and CDK8.16. A method in accordance with claim 15, wherein said protein kinase isa member selected from the group consisting of CDK1 and CDK5.
 17. Amethod of treating a cellular proliferative disease, said methodcomprising administering to a mammal having said disease atherapeutically effective amount of a compound of claim
 1. 18. A methodof inhibiting the growth of a tumor cell, said method comprisingcontacting said tumor cell with a compound of claim
 1. 19. A method inaccordance with claim 18 wherein said tumor cell is selected from thegroup consisting of lung, colon, breast, ovarian, prostate and hepaticcells.
 20. The method in accordance with claim 18 wherein said tumorcell is in a mammalian subject.
 21. The method in accordance with claim18 wherein said compound is formulated in a pharmaceutically acceptableform with an excipient or carrier.
 22. The method in accordance withclaim 18 wherein said compound is administered orally.
 23. The method inaccordance with claim 18 further comprising the step of observing for areduction in the growth of said tumor cell.
 24. A method of inhibitingthe growth of a tumor cell in a mammalian subject, said methodcomprising administering to said subject a therapeutically effectiveamount of a compound of claim
 1. 25. A method of treating aneurodegenerative disease, said method comprising administering to amammal having said disease a therapeutically effective amount of acompound of claim 1.